Cytomegalovirus (CMV) seropositivity is highly prevalent among both organ donors and recipients. Under life-long immunosuppression, post-transplant CMV disease is a significant contributor to poor graft and recipient outcome. Transplant tolerance allows complete avoidance of immunosuppression and is now clinically achievable. In such tolerant hosts, how the course of CMV infection may be modified has not been characterized. Conversely, how concurrent CMV infection may alter the outcome of tolerance is also largely unknown. In this application, we propose to examine the reciprocal interaction between CMV infection and transplant tolerance, using a highly clinically relevant murine model of transplant tolerance. In this model, donor-specific tolerance is achieved by pre-transplant delivery of donor negative vaccination consisting of donor cells treated with a crosslinker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). Our murine studies have already led to ongoing experiments in non-human primates, demonstrating promising efficacy of this strategy for both allogeneic and xenogeneic tolerance induction. More importantly, our colleagues recently published a first-in-man clinical trial using ECDI-fixed peptide-coupled autologous cells for multiple sclerosis, establishing the feasibility, safety and efficacy of this novel tolerance strategy. Using murine CMV in this model, we demonstrate that both acute and latent CMV infection impair tolerance induction and destabilize established tolerance, likely via interfering with Receptor Tyrosine Kinase (RTK)-mediated efferocytosis and directly or indirectly interfering with expansion of host myeloid derived suppressor cells (MDSCs). Conversely, presence of donor-specific tolerance in the host suppresses immediate early (IE) gene transcription necessary for latent CMV reactivation. Based on these findings, we hypothesize that CMV infection impairs tolerance via disrupting RTK-mediated tolerogenic interaction between host cells and ECDI-fixed donor cells, and conversely tolerance inhibits CMV reactivation via inhibiting transplant-induced inflammation, an obligatory early triggers for CMV genome epigenetic reprogramming. In this application, we propose to examine: (1) the effects and mechanisms of CMV-induced tolerance impairment; (2) the effects and mechanisms of tolerance-induced inhibition of CMV reactivation. Our long-term goal is to define targeted therapies for establishing and maintaining stable tolerance in hosts with CMV infection.